Na-HCO3 Cotransporter Research Articles

CAMP-dependent secretagogues stimulate the NaHCO3 cotransporter in the villous epithelium of the brushtail possum, Trichosurus vulpecula.

In the ileum of the brushtail possum, Trichosurus vulpecula, fluid secretion appears to be driven by electrogenic HCO3- secretion. Consistent with this, the cystic fibrosis transmembrane conductance regulator is expressed in the apical membrane of the ileal epithelial cells and the pancreatic or secretory variant of the NaHCO3 cotransporter in the basolateral membrane. This suggests that in the possum ileum, electrogenic HCO3- secretion is driven by basolateral NaHCO3 cotransporter (NBC) activity. To determine if the NBC contributes to HCO3- secretion in the possum ileum, intracellular pH (pHi) measurements in isolated villi were used to demonstrate NBC activity in the ileal epithelial cells and investigate the effect of cAMP-dependent secretagogues. In CO2/HCO3--free solutions, recovery of the epithelial cells from an acid load was Na+-dependent and ≈80% inhibited by ethyl-isopropyl-amiloride (EIPA, 10µmol L-1), indicative of the presence of an Na+/H+ exchanger, most likely NHE1. However, in the presence of CO2/HCO3-, EIPA only inhibited ≈ 50% of the recovery, the remainder was inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS, 500µmol L-1), indicative of NBC activity. Under steady-state conditions, NHE1 inhibition by EIPA had little effect on pHi in the presence or absence of secretagogues, but NBC inhibition with DIDS resulted in a rapid acidification of the cells, which was increased fivefold by secretagogues. These data demonstrate the functional activity of an NaHCO3 cotransporter in the ileal epithelial cells. Furthermore, the stimulation of NBC activity by secretagogues is consistent with the involvement of an NaHCO3 cotransporter in electrogenic HCO3- secretion.

Hypertension in NBCe2 Deficient Mice Caused by Enhanced ENaC Activity

Mutations of the electrogenic Na‐HCO3 cotransporter NBCe2 are associated with hypertension but the detailed pathogenesis is not well understood. Here we showed that up‐regulation of the epithelial Na channel (ENaC) in the distal nephron plays a critical role in hypertension caused by NBCe2 deficiency in mice. Wild type (WT) and NBCe2 knock out (KO) mice were fed an acid diet (1.5% NH4Cl) for 7‐10 days. Mean arterial pressure (MAP) was measured by tail cuff before and after the treatment with amiloride (Amil, 25 mg/L in drinking water). The MAP (mmHg) was higher in KO compared with WT before treatment (KO: 124.5 ± 2.2 vs WT: 110.6 ± 4.4). After treatment, the MAP in both groups dropped to a similar level (KO: 98.4 ± 3.7 vs WT: 99.1 ± 4.4). Western blot showed that the ENaC‐α expression in the kidney cortex was higher in KO compared with WT. Metabolic cage experiments showed that urinary Na excretion increased 3‐fold after Amil treatment (5 mg/kg, ip) in KO, while increasing only 20% in WT. The urinary K excretion also decreased significantly after Amil treatment in the KO but was similar before and after treatment in WT. Micropunture experiments showed that the [K] was significantly higher in the connecting tubule (CNT) of the KO compared with WT (KO: 21.9 ± 0.6 vs WT: 17.9 ± 1.5 mM), while the transepithelial potential (Vte, mV) was significantly lower (KO: ‐3.2 ± 0.2 vs WT: ‐1.6 ± 0.2). After Amil treatment, the Vte increased to ‐0.1 ± 0.2 in KO and 0.7 ± 0.4 in WT. These results show that both the ENaC expression and activity are increased in the KO, which contribute to the hypertension caused by NBCe2 deficiency.

Inflammation reduces the activity of the NaHCO3 cotransporter, NBCe1, in the surface cells of the proximal colon of IL10 knockout mice (908.1)

The impact of inflammation on intestinal secretion is contentious. Here the effect of inflammation on activity of the NaHCO3 cotransporter, NBCe1, in the proximal colon was investigated. Interleukin10‐knockout (IL10‐/‐) mice infected with Helicobacter typhlonius, which develop colitis, were used as a model of inflammation, and uninfected, asymptomatic IL10‐/‐ mice served as controls. NBCe1 activity was measured as the 5‐(N‐ethyl‐N‐isopropyl) amiloride‐insensitive (EIPA, 20µM), 4,4'‐diisothiocyano‐2,2'‐stilbenedisulfonic acid‐sensitive (DIDS, 500µM), rate of intracellular pH recovery and acid extrusion (JH+) following acid loading of isolated crypts, while NBCe1 expression was measured by Western blotting and immunohistochemistry. In control tissues NBCe1 immunoreactivity was restricted to the surface cells and upper third of the crypts. Consistent with this there was little evidence of NBCe1 activity in the lower half of the crypts, with or without forskolin (10µM) stimulation. In contrast, in the surface cells, while NBCe1 was inactive under basal conditions (JH+EIPA=16.4±1.8mM.min‐1; JH+EIPA+DIDS=18.7±1.8 mM.min‐1), its activity was stimulated by forskolin (after forskolin JH+EIPA=24.5±2.7 mM.min‐1 and JH+EIPA+DIDS=13.6±1.1mM.min‐1). In inflamed tissues, total NBCe1 expression was reduced (P<0.001, n=6), there was no NBCe1 immunoreactivity in the surface cells and upper crypts, and forskolin did not stimulate NBCe1 activity in the surface cells. This suggests inflammation reduces colonic HCO3‐ secretion, which may affect luminal pH regulation and growth of luminal bacteria, possibly contributing to the dysbiosis seen in patients with inflammatory bowel disease, as well as modifying mucus hydration.Grant Funding Source: Supported by The Otago Medical Research Foundation

Direct evidence for calcineurin binding to the exon-7 loop of the sodium-bicarbonate cotransporter NBCn1.

The NaHCO3 cotransporter NBCn1 plays a role in neutralizing intracellular acid loads at the basolateral membrane in cells of the medullary thick ascending limb (mTAL). Calcineurin inhibitors (Cn-Is) are known to both downregulate NBCn1 expression in the distal nephron and cause renal tubular acidosis (RTA), a risk factor for nephrocalcinosis and nephrolithiasis. In this report, we provide a new perspective on concurrent studies of NBCn1 in various tissues by using cell-free binding assays to investigate the interaction of NBCn1 with the calcineurin (Cn) isoform PPP3CA. Surface plasmon resonance (SPR) analyses show that the protein domain Exon 7 (translated from cassette II of NBCn1) binds Cn with an equilibrium dissociation constant (KD) of 30 +/- 15 nm. Linked-reaction tests suggest that the binding involves a conformational change. Nested PCR reactions were used to show that NBCn1-Exon 7 splice variants with alternative N-termini regions are expressed in the kidney, as well as other tissues. Additionally, we discuss NBCn1-Exon 7 implication in acid-base balance and calcium crystallization in the kidney.

Identification of Functionally Distinct Na-HCO3 Co-Transporters in Colon

Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5′-diisothiocyanato-2-2′-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H+]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in pHi regulation in colonic epithelial cells.

Metabolic Communication between Astrocytes and Neurons via Bicarbonate-Responsive Soluble Adenylyl Cyclase

Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO₃⁻) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response to HCO₃⁻ entry via the electrogenic NaHCO₃ cotransporter (NBC). Activated sAC increases intracellular cAMP levels, causing glycogen breakdown, enhanced glycolysis, and the release of lactate into the extracellular space, which is subsequently taken up by neurons for use as an energy substrate. This process is recruited over a broad physiological range of [K⁺](ext) and also during aglycemic episodes, helping to maintain synaptic function. These data reveal a molecular pathway in astrocytes that is responsible for brain metabolic coupling to neurons.

Corrigendum

CorrigendaCorrigendumPublished Online:15 Mar 2012https://doi.org/10.1152/ajpcell.zh0-6921-corr.2012Original articleMoreSectionsPDF (30 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations Volume 302, February 2012Volume 71, February 2012Lee SK, Boron WF, Parker MD. Am J Physiol Cell Physiol 302: , 2012. First published October 19, 2011; doi:10.1152/ajpcell.00352.2011 (http://ajpcell.physiology.org/content/302/3/C518).—The title of the article was printed incorrectly and should read as follows: “Relief of autoinhibition of the electrogenic Na-HCO3 cotransporter NBCe1-B: role of IRBIT vs. amino-terminal truncation.” The title has been corrected online.This article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformation Related ArticlesRelief of autoinhibition of the electrogenic Na-HCO3 cotransporter NBCe1-B: role of IRBIT vs. amino-terminal truncation 01 Feb 2012American Journal of Physiology-Cell Physiology More from this issue > Volume 302Issue 6March 2012Pages C941-C941 Copyright & PermissionsCopyright © 2012 the American Physiological Societyhttps://doi.org/10.1152/ajpcell.zh0-6921-corr.2012History Published online 15 March 2012 Published in print 15 March 2012 Metrics

The NaHCO3 cotransporter in the possum ileum is stimulated by secretagogues

The NaHCO3 cotransporter in the possum ileum is stimulated by secretagogues

Activation and Inactivation Mechanisms of Na-HCO3 Cotransporter NBC1

The electrogenic Na + -HCO3 - cotransporter NBC1, also known as NBCe1, is expressed in several tissues such as kidney, pancreas, eye, and brain. NBC1 has three variants, which may mediate different physiological functions. Inacti- vating mutations in NBC1 result in proximal renal tubular acidosis associated with ocular abnormalities. Functional analysis suggests that at least 50% reduction in NBC1 activity is required to induce severe acidemia invariably associated with stunted growth. Some mutations in the NBC1 gene may also induce trafficking abnormalities. On the other hand, the variant-specific N-terminal regions seem to play an essential role in the full activation of NBC1. This review will focus on the recent progress in physiology and pathophysiology of NBC1.

Multiplicity of cerebrospinal fluid functions: New challenges in health and disease.

This review integrates eight aspects of cerebrospinal fluid (CSF) circulatory dynamics: formation rate, pressure, flow, volume, turnover rate, composition, recycling and reabsorption. Novel ways to modulate CSF formation emanate from recent analyses of choroid plexus transcription factors (E2F5), ion transporters (NaHCO3 cotransport), transport enzymes (isoforms of carbonic anhydrase), aquaporin 1 regulation, and plasticity of receptors for fluid-regulating neuropeptides. A greater appreciation of CSF pressure (CSFP) is being generated by fresh insights on peptidergic regulatory servomechanisms, the role of dysfunctional ependyma and circumventricular organs in causing congenital hydrocephalus, and the clinical use of algorithms to delineate CSFP waveforms for diagnostic and prognostic utility. Increasing attention focuses on CSF flow: how it impacts cerebral metabolism and hemodynamics, neural stem cell progression in the subventricular zone, and catabolite/peptide clearance from the CNS. The pathophysiological significance of changes in CSF volume is assessed from the respective viewpoints of hemodynamics (choroid plexus blood flow and pulsatility), hydrodynamics (choroidal hypo- and hypersecretion) and neuroendocrine factors (i.e., coordinated regulation by atrial natriuretic peptide, arginine vasopressin and basic fibroblast growth factor). In aging, normal pressure hydrocephalus and Alzheimer's disease, the expanding CSF space reduces the CSF turnover rate, thus compromising the CSF sink action to clear harmful metabolites (e.g., amyloid) from the CNS. Dwindling CSF dynamics greatly harms the interstitial environment of neurons. Accordingly the altered CSF composition in neurodegenerative diseases and senescence, because of adverse effects on neural processes and cognition, needs more effective clinical management. CSF recycling between subarachnoid space, brain and ventricles promotes interstitial fluid (ISF) convection with both trophic and excretory benefits. Finally, CSF reabsorption via multiple pathways (olfactory and spinal arachnoidal bulk flow) is likely complemented by fluid clearance across capillary walls (aquaporin 4) and arachnoid villi when CSFP and fluid retention are markedly elevated. A model is presented that links CSF and ISF homeostasis to coordinated fluxes of water and solutes at both the blood-CSF and blood-brain transport interfaces.Outline1 Overview2 CSF formation2.1 Transcription factors2.2 Ion transporters2.3 Enzymes that modulate transport2.4 Aquaporins or water channels2.5 Receptors for neuropeptides3 CSF pressure3.1 Servomechanism regulatory hypothesis3.2 Ontogeny of CSF pressure generation3.3 Congenital hydrocephalus and periventricular regions3.4 Brain response to elevated CSF pressure3.5 Advances in measuring CSF waveforms4 CSF flow4.1 CSF flow and brain metabolism4.2 Flow effects on fetal germinal matrix4.3 Decreasing CSF flow in aging CNS4.4 Refinement of non-invasive flow measurements5 CSF volume5.1 Hemodynamic factors5.2 Hydrodynamic factors5.3 Neuroendocrine factors6 CSF turnover rate6.1 Adverse effect of ventriculomegaly6.2 Attenuated CSF sink action7 CSF composition7.1 Kidney-like action of CP-CSF system7.2 Altered CSF biochemistry in aging and disease7.3 Importance of clearance transport7.4 Therapeutic manipulation of composition8 CSF recycling in relation to ISF dynamics8.1 CSF exchange with brain interstitium8.2 Components of ISF movement in brain8.3 Compromised ISF/CSF dynamics and amyloid retention9 CSF reabsorption9.1 Arachnoidal outflow resistance9.2 Arachnoid villi vs. olfactory drainage routes9.3 Fluid reabsorption along spinal nerves9.4 Reabsorption across capillary aquaporin channels10 Developing translationally effective models for restoring CSF balance11 Conclusion

Arachidonic Acid Metabolites Inhibit the Stimulatory Effect of Angiotensin II in Renal Proximal Tubules

Angiotensin II (Ang II) regulates renal proximal transport in a biphasic way via Ang II type 1 receptor (AT1). Whereas extracellular signal-regulated kinase (ERK) activation mediates the stimulatory effect, cytosolic phospholipase A2 (cPLA2) mediates the inhibitory effect independently of ERK. In this study, we tested the hypothesis that the cPLA2/P450 epoxygenase pathway might work to suppress the Ang II-mediated ERK activation. In the presence of arachidonic acid or 5,6-epoxyeicosatrienoic acid (EET), Ang II failed to stimulate the Na-HCO3 cotransporter activity in renal proximal tubules isolated from wild-type, AT1A-deficient, and cPLA2-alpha-deficient mice. In addition, Ang II failed to induce a significant ERK phosphorylation in the presence of arachidonic acid or 5,6-EET. Arachidonic acid or 5,6-EET also suppressed the stimulatory effect of Ang II on net proximal tubule bicarbonate absorption without changing cell Ca2+ concentrations. These results indicate that the cPLA2-alpha/P450/EET pathway blocks the stimulatory effect of Ang II by suppressing the ERK activation. Thus, the cPLA2-alpha/P450/EET pathway may operate as a unique negative feedback mechanism to attenuate excessive Ang II activity in the renal proximal tubules, where extremely high concentrations of Ang II are found.

Physiology and pathophysiology of Na-HCO3 cotransporter NBC1

Physiology and pathophysiology of Na-HCO3 cotransporter NBC1

IRBIT functionally enhances the electroneutral Na + ‐coupled bicarbonate transporter NCBE by sequestering an N‐terminal autoinhibitory domain

IRBIT binds to and functionally enhances the electrogenic Na-HCO3 cotransporter NBCe1-B (Shirakabe et al., PNAS, 103, 2006). In an accompanying abstract, we further demonstrate that IRBIT functionally enhances the electroneutral Na-coupled HCO3 transporters NBCn1, NDCBE and NCBE. Taking the NCBE/IRBIT pair as our model, we co-expressed NCBE with a C-terminal EGFP tag (NCBE-EGFP) and IRBIT with an N-terminal HA-tag (HA-IRBIT) in Xenopus oocytes. The interaction was confirmed in oocytes by co-immunoprecipitation of HA-IRBIT (but not the S71A mutant known not to bind to NBCe1-B) with an anti-GFP antibody. We assayed the function of NCBE-EGFP by exposing oocytes to a CO2/HCO3 solution and monitoring the rate of the subsequent pHi recovery from the CO2-induced acid-load using pH-sensitive microelectrodes. NCBE-EGFP activity was enhanced by co-expression of HA-IRBIT but not the S71A mutant. Truncating the N terminus of NCBE by removing protein encoded by exons 1 (residues 1-16) or exons 1 and 2 (residues 1-43), affected neither NCBE-EGFP activity nor its enhancement by co-expressed HA-IRBIT. Removing the protein encoded by exons 1, 2 and 3 (residues 1-92) doubled the rate of pHi recovery—compared to full-length NCBE in the absence of co-expressed HA-IRBIT—and negated the enhancement in the presence of HA-IRBIT. Our data suggest that exon 3 of NCBE encodes an autoinhibitory domain (AID), and that IRBIT enhances the activity of NCBE by sequestering the AID.

Phospholemman and the Cardiac Sodium Pump

See related article, pages 1376–1383 In excitable tissues, the activity of the plasmalemmal sodium/potassium ATPase (Na/K pump) is vital for the maintenance of normal electrical activity and ion gradients. In cardiac muscle, the transsarcolemmal sodium (Na) gradient established by the Na/K activity is essential not only for generating the rapid upstroke of the action potential but also for driving a number of ion exchange and transport processes that are crucial for normal cellular function, excitation contraction coupling, ion homeostasis and the control of cell volume. These Na-dependent membrane transporters include those responsible for the regulation of other ions (such as the sodium calcium exchanger (NCX), Na/H exchanger and Na-HCO3 cotransporter), as well as those involved in the movement of substrates and amino acids.1 By determining the set point for NCX, the Na/K pump controls the predominant mechanism of transmembrane calcium (Ca) flux, and hence indirectly controls intracellular Ca load and myocardial contractility. Interventions that influence either the activity of the Na/K pump, or indirectly the transmembrane sodium gradient, can therefore profoundly affect normal cardiac function. Phospholemman, a type 1 transmembrane protein, is the predominant quantitative site of phosphorylation by protein kinase A (PKA) and protein kinase C (PKC) in cardiac sarcolemma.2 PKA phosphorylates serine 68 and PKC phosphorylates serines 63 and 68 in phospholemman. For a long time following its cloning in 1991,3 the physiological role of phospholemman was unclear. Indeed, writing in this journal in 1998, researchers noted that “As a major target for hormone-stimulated phosphorylation in the heart, the physiological function of phospholemman is likely to be an important one”,4 however they were unable to suggest what this function may be. Various roles have now been proposed: 1) regulation of cell volume; …

An alternate pathway of cAMP-stimulated Cl− secretion across the NKCC1-null murine duodenum

Background & Aims: Adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated anion secretion across the duodenal epithelium requires the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane and anion uptake proteins in the basolateral membrane. NKCC1, the epithelial Na+/K+/2Cl− cotransporter, is the major protein responsible for Cl− uptake. In this study, we evaluate the role of NKCC1 in determining the relative rates of transepithelial Cl− and HCO3− secretion during cAMP stimulation of the duodenum. Methods: Bicarbonate and chloride secretion across duodenal mucosa was measured in Ussing chambers by pH stat and 36Cl flux methods using mice with either gene-targeted deletion of NKCC1 (NKCC1−/−) or bumetanide blockade of NKCC1. Results: Total anion secretion stimulated by forskolin treatment of NKCC1-null duodenum resulted from approximately equivalent rates of electrogenic chloride, electrogenic bicarbonate, and electroneutral bicarbonate secretion. Evaluation of the alternate chloride secretory pathway indicated chloride uptake by a basolateral membrane anion exchange process with characteristics consistent with the anion exchanger isoform AE2. Conclusions: Chloride uptake by basolateral anion exchanger activity (AE2) supports intracellular cAMP–stimulated chloride secretion in the NKCC1-null duodenum. A model for the alternate chloride secretion pathway is proposed whereby chloride uptake via AE2 is coupled to basolateral NaHCO3 cotransport to support CFTR-mediated chloride and bicarbonate secretion.GASTROENTEROLOGY 2002;123:531-541

Characterization and molecular localization of anion transporters in colonic epithelial cells.

This study describes the identification and characterization of anion transporters in apical membrane (APM) and basolateral membrane (BLM) of rat distal colon. Cl-HCO3, Cl-OH, Cl-butyrate, and butyrate-HCO3 exchanges and Na-HCO3 cotransporter are present in rat distal epithelial cells. Cl-HCO3 exchange (1) is present only in APM from surface, but not from crypt cells; (2) is also present in BLM; and (3) of surface cell is encoded by anion exchange (AE)-1 isoform, whereas BLM Cl-HCO3 is encoded by AE2 isoform. Cl-OH exchange is present only in APM, but not in BLM from surface and crypt cells, and is responsible for regulation of cell functions (i.e., cell pH and cell volume regulation). Butyrate-HCO3 exchange (1) is also present in apical membrane vesicles (AMV) from surface, but not from crypt cells; (2) is present in BLM; and (3) is responsible for SCFA-dependent HCO3 secretion. By contrast, Cl-butyrate exchange: (1) is present in APM from both surface and crypt cells; (2) is not present in BLM; and (3) recycles butyrate by absorbing Cl. Na-HCO3 cotransport: (1) is present only in BLM; (2) is expressed predominantly in midcrypt regions; and (3) may be linked to HCO3 secretion. A mechanism for HCO3 movement across the crypt apical membrane has not as yet been identified.

Immunolocalization of the Na-HCO3 cotransporter (NBC) in rat gastric parietal cells

Immunolocalization of the Na-HCO3 cotransporter (NBC) in rat gastric parietal cells

Regulation of renal Na-HCO3 cotransporter: VIII. Mechanism Of stimulatory effect of respiratory acidosis.

We examined the effect of respiratory acidosis on the Na-HCO3 cotransporter activity in primary cultures of the proximal tubule of the rabbit exposed to 10% CO2 for 5 min, 2, 4, 24 and 48 hr. Cells exposed to 10% CO2 showed a significant increase in Na-HCO3 cotransporter activity (expressed as % of control levels, 5 min: 142 +/- 6, 2 hr: 144 +/- 13, 4 hr: 145 +/- 11, 24 hr: 150 +/- 15, 48 hr: 162 +/- 24). The increase in activity was reversible after 48 hr. The role of protein kinase C (PKC) on the stimulatory effect of respiratory acidosis on the cotransporter was examined in presence of PKC inhibitor calphostin C or in presence of PKC depletion. Both calphostin C and PKC depletion prevented the effect of 10% CO2 for 5 min or 4 hr to increase the activity of the cotransporter. 10% CO2 for 5 min or 4 hr increased total and particulate fraction PKC activity. To examine the role of phosphotyrosine kinase (PTK) on the increase in cotransporter activity we studied the effect of two different inhibitors, 2-hydroxy-5-(2,5-dihydroxylbenzyl) aminobenzoic acid (HAC) and methyl 2,5-dihydroxycinnamate (DHC) which inhibit phosphotyrosine kinase in basolateral membranes. Cells were pretreated either with vehicle or HAC or DHC and then exposed to 10% CO2 for 5 min or 4 hr. In cells treated with vehicle, 10% CO2 significantly increased cotransporter activity as compared to control cells exposed to 5% CO2. This stimulation by 10% CO2 was completely prevented by HAC or DHC at 5 min (5% CO2: 1.8 +/- 0.2, 10% CO2: 2.6 +/- 0.2, 10% CO2 + HAC: 1.6 +/- 0.2, 10% CO2: +DHC: 2.0 +/- 0.3 pH unit/min) and also at 4 hr. The protein synthesis inhibitors actinomycin D and cycloheximide appear to prevent the effect of 10% CO2 for 4 hr on the cotransporter. Our results show that early respiratory acidosis stimulates the Na-HCO3 cotransporter through PKC and PTK-dependent mechanisms and the late effect appears to be mediated through protein synthesis.

Apical and basolateral membrane mechanisms that regulate pHi in bovine retinal pigment epithelium.

pH regulation was studied in fresh explant bovine retinal pigment epithelium-choroid using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and intracellular microelectrodes. Acid recovery was HCO3 dependent, inhibited by apical amiloride and apical or basal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and required apical and basal Na. Alkali recovery was HCO3 dependent and inhibitable by apical or basal DIDS. Three apical and two basolateral transporters were identified. Four contribute to acid extrusion, i.e., apical Na/H exchange, apical H-lactate cotransport, and apical Na-HCO3 cotransport and basolateral Na-HCO3 cotransport. At least two contribute to alkali extrusion, i.e., apical Na-HCO3 cotransport and a basolateral HCO3-dependent, DIDS-inhibitable mechanism, possibly Na-HCO3 cotransport, Cl/HCO3 exchange, or both. The apical Na-HCO3 cotransporter is electrogenic, carrying net negative charge inward. Basal Cl removal or addition of basal HCO3 caused HCO3- and Cl-dependent alkalinizations, respectively. Apical DIDS increased both responses. These cytosolic pH (pHi) regulatory mechanisms are so tightly coupled that changes in pHi can only occur after two or more of them are inhibited. In addition, these mechanisms help provide pathways for transport of Na and HCO3 across the retinal pigment epithelium between the blood and the distal retina.

Regulation of the renal Na-HCO3 cotransporter: VII. Mechanism of the cholinergic stimulation

Cholinergic agents regulate proximal tubule acidification but the mechanism responsible for this effect is unclear. We examined the effect of the cholinergic agent carbachol on the activity of the Na-HCO3 cotransporter in primary cultures of the proximal tubule of the rabbit. The activity of the cotransporter was assayed either as HCO3-dependent 22Na uptake or as the recovery of intracellular pH in cells perfused continuously with Cl-free physiologic solution containing amiloride to block the Na-H antiporter. Carbachol caused a dose-dependent stimulation of the cotransporter activity with a maximum increase of 90% above control values at 10(-5) M and half maximal stimulation at 10(-7) M. The stimulation was blocked by atropine and pirenzepine indicating an effect through the M1 muscarinic receptor. Carbachol increased intracellular calcium fourfold and the rise in cytosolic calcium was prevented by the intracellular calcium chelator, BAPTA. BAPTA also blocked the effect of carbachol on the cotransporter. Because carbachol activates phospholipase C and protein kinase C, we examined the effect of carbachol in the presence of the phospholipase C inhibitor, U73122, or the PKC inhibitor, calphostin C, or PKC depletion. The phospholipase C inhibitor prevented both the effect of carbachol on the cotransporter and on the intracellular Ca. Calphostin C and PKC depletion also prevented the stimulation of the cotransporter. Carbachol increased PKC activity and caused translocation of the PKC to the particulate fraction. We also examined the effect of the phosphatase inhibitor, calyculin A or the calmodulin kinase inhibitor, W-13 on carbachol stimulation. Calyculin A and W13 likewise prevented the carbachol-induced stimulation of the cotransporter. These results demonstrate that cholinergic stimulation modulated the activity of the cotransporter through multiple pathways including phospholipase C/PKC and phosphatase systems.